in situ hybridization and RNA probe design - (Sep/07/2005 )
Recently, i'am asked to do RNA ISH experiment in our lab.But i am a new comer so i know little about it. i use the pGEM-TEasy vector to synthesis sense and antisense probes in vitro.you kown the target PCR product insert to the vector through TA clone, and the insert site is fixed which is the Ecol Ⅴrestriction enzymatic site. when i blasting the sense and antisense probes in internet, i found approxi 30bp can match with other RNA in arabidopsis thaliana which is not the sequence of the part of target cDNA, but part of the vector sequnces from the SP6 promotor to the insert site which totally has 83bp. Whether there is someone know how should i do, please tell me! Will the 30bp sequence seriously affect the consequnce? if it do, how many basepairs match is permitted? is it neccesary for me use other vecors instead of pGEM-TEasy vector?
longing for your replies!
any help or advise is much appreciated!
longing for your replies!
any help or advise is much appreciated!
As far as we know, the more nonspecific sequence of probe match with other RNA, the more terrible consequnce i will have, in that the background will be high, and also can appear pseudo positve signal
who could do me a favour?
please give me some advises!
looking forward to your replies!
Sorry I can not help U.
We are doing FISH with RNA probes, but in Archaea
You look desperate
Hope you find something but post something in
http://www.microbial-ecology.net/probebase/ may be someone will help you
Keep fighting!!!
hi
for designing the probes you mentionned, you can use the genscript siRNA designer which allows a blast against human, mouse, rat, drosophila, c elegans, xenopus laevi, zebrafish, bovine, chicken, and arabidopsis genomes. It will help avoid other complementaries.
30bp is theorically sufficent to get an hybridization. can you tell how long is your probe?
But you can minimize it as these 30bp are a small part of the total probe by increasing the stringency of hybridization/wash steps (higher temp, more salts...)
Fred
for designing the probes you mentionned, you can use the genscript siRNA designer which allows a blast against human, mouse, rat, drosophila, c elegans, xenopus laevi, zebrafish, bovine, chicken, and arabidopsis genomes. It will help avoid other complementaries.
30bp is theorically sufficent to get an hybridization. can you tell how long is your probe?
But you can minimize it as these 30bp are a small part of the total probe by increasing the stringency of hybridization/wash steps (higher temp, more salts...)
Fred
Thank you for giving me help! fortunately,i have already successfully coped with my problems. I alter a restriction enzyme in the right border of the insert site to minimize the sequence which is not neccessary.
Thank you all the same!